SeHed, a novel gene expression system with stress-evoked hydrogen peroxide elimination property and anti-aging effect

moderate of to cellular functions such as proliferation, differentiation, and infection resistance, but the excessive level of ROS causes oxidative damage which underlies the basic mechanism for aging and geriatric diseases. 1 Therefore, precisely managing cellular ROS levels, meaning to keep redox homeostasis properly, stands as an aim for health and longevity. Actually, this aim is challenging, as it asks not only to restrain excessive ROS accumulation at the right time and right place but also to guarantee proper ROS levels for ﬁ tting physiological requirements. Traditional ways for eliminating ROS put more attention on the ef ﬁ ciency and identi ﬁ ed a variety of drugs and enzymes with antioxidant properties, known as enzymatic and non-enzymatic ROS scavengers, respectively. However, how to accurately manage the integrative effect of ROS scavengers in vivo remains a problem. Actually, the effectiveness of non-enzymatic antioxidants in vivo is controversial, explained mostly by the uncertainty to reach a given organ with the appropriate concentra-tion or to work there persistently. 2 As to enzymatic ROS scavengers, although their overexpression in a given place can be achieved, this mode is often unwieldy or harmful to intrinsic physiologic functions in cases. 3 For this reason, we attempt to develop new ways for in vivo antioxidation, for establishing a high-quality balance between ef ﬁ ciency and safety. As the consequence, we innovated a gene expression system that can limit the excessive ROS accumulation but keep the level of ROS.

supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2. P. aeruginosa wild-type (WT) strain PAO1 was obtained from Dr. Zhou (Sichuan university, Sichuan, China). The primary myoblasts from about 10-day-old C57BL/6J mice were isolated and cultured following the protocol of Xiaobo wang et al 1 . To induce myogenic differentiation, cells were grown to 60-70% confluence in growth media and then replaced the differentiation medium. Meticulously, primary myoblast cells were cultured in DMEM supplemented with 2% (v/v) horse serum for differentiation, and H9C2 cells were cultured in DMEM supplemented with 2% (v/v) horse serum plus 10 μM retinoic acid for differentiation.

Ethics and Animal procedures
The C57BL/6j mice were purchased from Beijing HFK Bioscience Co., Ltd. All mice were maintained on a 12/12 hours. Eight-week-old male mice were placed for 24 hours into cages and received food and water ad libitum. All animal procedures were performed following the protocol approved by the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, China, approval No. 2016064A).
Mice were tail-vein-injected with a dose of 5×10 11 GC per mouse AAV9-SeHed and AAV9-control in the experiment group and in the control group, respectively. one week later, the mice were injected with D-galactose for the progeria model or fed with highfat diet (60% fat, 20% protein calories, and 20% carbohydrate calories) for the metabolic disorder model. Meticulously, for D-galactose induced mice progeria model, mice were randomly divided into the Saline (Saline) group, D-galactose (D-gal) group and SeHed group. Saline and D-galactose group mice were injected with AAV9-control virus, and SeHed group mice were injected with AAV9-SeHed virus. One week after AAV injection, the Saline group mice were intraperitoneally injected with saline solution, and D-gal group and SeHed group mice were intraperitoneally injected with D-gal (100 mg/kg/day) for four months. For HFD induced mice metabolic disorder models. All mice were randomly divided into SD group, HFD group and SeHed group.
SD and HFD group mice were injected with AAV9-control virus, and SeHed group mice were injected with AAV9-SeHed virus. One week after AAV injection, SD group mice were fed a standard diet (SD; 10% fat), HFD and SeHed group mice were fed a High-Fat diet (HFD; 60% fat) for four months.

Cellular senescence models
H2O2-induced cellular senescence model: a modified H2O2 treatment protocol was used. NIH3T3 was trypsinized and suspended in phosphate buffer solution (PBS) at a density of 1×10 6 cells/mL. The cell suspension was transferred to an Eppendorf tube, and then the cells were exposed to 400 μM H2O2, incubating at 37℃ for 45 min 2 . Then, the treated cells were then seeded to plate for indicated experiments, and the cells had undergone different treatments as described in individual figure legends. For H2O2 induced MRC-5 senescence. MRC-5 were seeded at a 12/24 well culture plates, after cells were cultured for 12 hours, then cells were exposed to PBS contain 400 μM H2O2, incubating at 37℃ for 60 min. Then, removing PBS, adding complete medium and continue to culture for 3 days. For drugs (Adriamycin, Etoposides, or Rotenone) induced cellular senescence model 3,4,5 . NIH3T3 or MRC-5 were seeded at a 12/24 well culture plates. After culture for 24 hours, the cells were treated with drugs (0.5 μM Adriamycin or 5 μM Etoposides or 0.5 μM Rotenone for 2 days, and continue to culture for 5 days and analyzed later.

Senescence-associated β-galactosidase (SA-β-Gal) staining
For SA-β-Gal staining of cells in vitro. Intracellular SA-β-Gal activity was assayed using SA-β-Gal staining kit (Solarbio, Beijing, China, Cat No, G1580) according to the standard protocol, and senescent cells were identified as bluish green stained cells under a phase-contrast microscope. The percentage of β-Gal-positive cells in total cells was determined by counting 1000 cells in 7 random fields, for each group. The results were expressed as the mean ± SD.
For SA-β-Gal staining of tissues (Liver, Lung and Fat) in vivo. Tissues were fixed with 10% paraformaldehyde for 24 hours. Then, using gradient concentrations of sucrose for dehydration, and tissues was embedded in OCT, sectioned at 5 μm thickness. After rehydration in PBS, the SA-β-Gal staining was performed using SA-β-Gal staining kit as previous description (Liver incubated 12 hours, Lung incubated 24 hours). In addition, the fat tissue was cut into little pieces, and the SA-β-Gal staining were performed with 12 well culture plate in the staining solution at 37°C for 8-10 hours.

Intracellular ROS, mROS and H2O2 level detection
Cellular ROS level was labeled by ROS probe DCFH-DA probe (Applygen, C1300) and detected by fluorescence microscope or flow cytometry. DCFH-DA turns into a green fluorescent molecule called DCF when oxidized by ROS, intracellular ROS levels can be reflected by the fluorescence intensity of DCF 6 . Three independent experiments were conducted. Cellular H2O2 level were analyzed by protein probe-HyperRed 8 . NIH3T3 was stably expressing HyperRed and SeHed plasmid mediated by lentivirus system. Then, these cells were plated on cover-glass in 2 cm-dishes (Nest, 801001). After cell senescence induced by H2O2, the medium was subsequently replaced with 2 ml of phenol-red-free DMEM supplemented with 10% FBS. The cultured dishes were placed in a 37 °C chamber equilibrated with humidified air containing 5% CO2 throughout the microscopy experiment. The fluorescence signals of HyperRed was recorded (excitation at 561 nm, detection at 620 nm) by confocal microscopy. All images were processed using Image J (fiji-win64; National Institutes of Health, Bethesda, MD, USA).

Double luciferase assay and EGFP report assay
For double luciferase assay, 1×10 4 -3×10 4 HEK293T cells were seeded in 2 ml of growth medium into 24-well plates and incubated under standard cell culture conditions for 24 hours. Then, cells were co-transfected with pTR-L and pGL3B-NF8p as described previously using Neofect™ DNA transfection reagent according to manufacturer's recommendations. After incubation for 24 hours, cells were treated with 100 μM H2O2 or treatment. Following 8-10 hours drug exposure, proteins were isolated and luciferase signals were analyzed using the Dual-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. Others, as to siRNA experiment, cells were transfected by siRNA (sip65, (sense, 5′-GCAUGCGAUUCCGCUAUAATT-3′ and antisense, 5′-UUAUAGCGGAAUCG CAUGCTT-3′) was obtained from Sangon Biotech (Shanghai, China)) and co-transfected with pTR-L and pGL3B-NF8p as described previously using Neofect™ DNA transfection reagent according to manufacturer's recommendations. After cells were incubated for 24 hours, cells were treated with 100 μM H2O2 or 10 ng/ml TNFα for 8-10 hours. Proteins were isolated and luciferase signals were analyzed using the Dual-Glo Luciferase Assay System (Promega) according to manufacturer's instructions.
For EGFP report assay, 1×10 5 HEK293T cells were seeded in 1 ml of growth medium into 12-well plates and incubated under standard cell culture conditions for 24 hours. Then, cells were transfected with pNF8p-EGFP as described previously using Neofect™ DNA transfection reagent according to manufacturer's recommendations.
After incubation for 24 hours, cells were treated with 100 μM and 200 μM H2O2, 0.5 μM ADR or 10 ng/ml TNFα for 24 hours. The EGFP signals were recorded by fluorescence microscope (Olympus, Tokyo, Japan) and analyzed by Image J software (fiji-win64; National Institutes of Health, Bethesda, MD, USA).

Plasmid construction
pGL3B-NF8p plasmid: The NF8p (promotor) fragment containing 8 NF-κB responsive elements is from document 9 . Firstly, the NF8p fragment was obtained by artificial synthesis way (from Tsingke Biotechnology Co., Ltd.). The PCR primers of We constructed the AAV-SeHed plasmid by recombinase for integrating the NF8p-mCat fragment into the Mlu I site of AAV empty vector. The HyperRed plasmid was donated by professor Chen zhouzao (Peking Union Medical College).

Plasmid transfection and lentiviral infection
For the expression of mCat, HEK293T cells were transfected with CMV-mCAT or SeHed respectively, Jet PRIME transfection reagents (PolyPlus, Illkirch, France) was

AAV production and purification
Triple-plasmid transfection using polyethylenimine (PEI, Polyscience) was carried out to produce the recombinant AAV. The transfer plasmids AAV-SeHed; pRep2CapX of AAV-X encoding Rep2 and CapX proteins plasmids; and pHelper were co-transfected into HEK293T cells. The cells were cultured in Dulbecco's modified essential medium (DMEM; Invitrogen, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% S/P antibiotics (Gibco, USA) at 37 °C. When the cells reached 80% confluence, they were transfected in 150 mm plates with 12 μg of pHelper plasmid, 10 μg of AAV pRep2CapX plasmids, and 6 μg of transfer AAV-SeHed plasmids for each plate. At 72 hours post transfection, cells were harvested by 4,000 g centrifugation at 4 °C for 30 min. The pellet was collected and re-suspended in buffer containing 10 mM Tris-HCl, pH 8.0. The suspension was subjected to four freeze-thaw cycles by dry ice/ethanol and a 37 °C water bath. The cell debris was sonicated and then digested with DNase I (200 units in 1.5 ml) for 1 hour at 37 °C. Following centrifugation at 10,000 g for 10 min at 4 °C, the supernatant was collected as the AAV crude lysate. The crude lysate was diluted with discontinuous gradient of 15%, 25%, 40% and 60% Iodixanol in a 39-ml ultracentrifuge tube (QuickSeal, 342414). After ultracentrifugation at 350,000 g and 18°C for 1 hour, 3 ml fractions at lower position of 40% and 0.5ml of 60% upper layer were collected, then repeat the ultracentrifugation at 350,000 g and 18°C for 1 hour one more time and desalted the fractions using a 100 kDa Cutoff Ultrafiltration tube (15 ml; Millipore, USA).
The purified AAV were stored at -80 °C before usage. The viral titers were determined by SYBR Green qPCR.

Living imaging
For the EGFP fluorescence imaging in vivo. we used the hair shaver for cutting off hair in the back or abdomen of mice. Then, all mice were anesthetized by isoflurane (RWD), and the EGFP fluorescence signals was collected by IVIS Lumina III (PerkinElmer) and the EGFP signals pictures were processed by IVIS Lumina III software.
For the PAO1 luminescence imaging in vivo. Due to the PAO1 has luminescent group. The PAO1 signals in vivo were directly collected by IVIS Lumina III after PAO1 were injected into the mice. The PAO1 luminescence signals pictures were processed by IVIS Lumina III software.

Fasting blood, IPGTT and IPITT
For fasting blood tests, blood glucose levels of all mice were detected via Roche glucometer after 16 hours of fasting. For glucose tolerance test (GTT), mice received one dose of glucose (1 g/kg body weight) via intraperitoneal injection after 16 hours of fasting, and blood glucose levels was measured at 0, 15, 30, 60, 90, and 120 min after glucose injection. For insulin tolerance test (ITT), mice were fasted for 4 hours and then intraperitoneal injected with insulin (0.75 units/kg body weight), and blood glucose levels was measured at 0, 15, 30, 60, 90, and 120 min after insulin injection.

Hematoxylin-Eosin staining Oil red O staining
Liver and Kidney were fixed in formalin, embedded in paraffin and sectioned for H&E staining. Liver sections were fixed in 10% formalin overnight. For the detection of lipid accumulation, frozen liver sections were stained with oil red O according to a standard protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Finally, liver sections were imaged at 200×magnification (Olympus, Tokyo, Japan). The area stained with oil red O solution was analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).

Exercise capacity, grip-strength test and Y-maze test
For mice exercise capacity test. All mice were acclimated to the treadmill system before running test. The exercise regimen commenced at a speed of 15 m/min with an inclination of 5 degrees. Mice were considered to be exhausted and removed from the treadmill following the accumulation of 10 or more shocks (0.5 mA) per minute for two consecutive minutes. The electric shock times were registered within 10 minutes running period.
For grip-strength test. A grip-strength test in mice was performed using a gripstrength meter. Mice were held in front of a horizontal bar, in such a way that only the forelimb paws could grasp the bar. To perform the test, animals were gently pulled back with a steady force until both paws released the bar. Peak tension in grams was recorded for three consecutive trials.
For Y-maze test. The three arms of the Y maze were randomly set as the start arm, the novel arm, and the other arm. Before each test, the novel arm was blocked with a partition, the mouse was put in from the starting arm, and allowed to move freely between starting arm and other arms to adapt for 10 minutes. After 1 hour, opened the new arm, put the mice in platform from the starting arm, let it move freely in the three arms for 5 minutes, the route of mice autonomous movement was recorded. After each test, the Y-maze platform was cleaned with 75% alcohol to remove odors and residues.
In the experiment, the starting arm, novel arm and other arms of different mice were randomly arranged. The calculation method is as follows: suppose that the new arm is A, the starting arm is B, the other arm is C, and the movement route of the mice during the test is BACABCBAB. During the test, the mice path passes through 9 arms, and the number of shuttles is 9; consecutive ABC, BCA, and CAB are counted as 1 point. In this example, there are BAC, CAB, ABC, and CBA, that is, the alternate score is 4. The spontaneous alternation score (%) = [(Number of alternations) / (Total arm entries-2)] *100. In this example, the alternating score rate % = [alternative score 4 / (total number of arms 9-2)] *100%=57.14%.

Biochemical evaluation
Alanine transaminase (ALT) and aspartate transaminase (AST) enzyme activity in serum were determined to evaluate liver injury. Urea and Creatinine levls in serum were determined to evaluate liver injury. Triglyceride and LDL level were evaluated for lipid metabolism. Above blood indexes were detected using a Cobas702 automatic biochemical analyzer (Roche Diagnostics GmbH) in Public Experimental Technology Center, West China Hospital, Sichuan University.

Infection experiments
PAO1 bacteria were grown overnight in lysogeny broth (LB medium) at 37°C with 220 rpm shaking. Next day, the bacteria were collected by centrifugation at 5000×g and resuspended in 20 ml of fresh LB medium, in which they were allowed to grow until the logarithmic phase. Then, the optical density (OD) at 600 nm was measured (1 OD =

Immunofluorescence
In brief, cells were fixed in 4% (v/v) paraformaldehyde in PBS for 15min at room temperature. Cells were washed thrice with PBS and permeabilized with 0.25% (v/v) Triton X-100 in PBS for 15 min. After washing thrice times with PBS, cells were blocked in 5% BSA in PBS for 45min at room temperature. Cells were then incubated with primary antibodies in 5% (w/v) BSA in PBS overnight at 4°C. The next day, the cells were washed thrice times with PBS, each for 10 min, followed by incubation with secondary antibody, in 5% (w/v) BSA/PBST for 1 hour at room temperature. The cells were then washed thrice times in PBS, incubated with 1μg/ml DAPI in PBS for 6 min, and washed twice with PBS. The slides were imaged by fluorescent confocal microscope. Cells with more than five visible spots at the expected location were Fusion Solo Imaging System. Every experiment was repeated at least 3 times, and representative data are shown.

qRT-PCR
Total RNA was isolated from cultured cells using Trizol (Takara, Shiga, Japan) and 1 µg of total RNA were used for reverse transcription by HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech co., ltd, Nanjing, China, Cat No. R223-01), and then quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green qPCR Master Mix (Biotool, Shanghai, China, Cat No, B21203). PCR reactions were performed in triplicate and the relative amount of cDNA was calculated by the comparative CT method using the 18s ribosomal RNA sequences as the control.
The primer sequences used for qRT-PCR are shown in supplementary Table 1.
Experiments were repeated three times.

Statistical analysis
Data are presented as means ± SD from at least 3 biological replicates. Statistical       a. The structure information of AAV9-SeHed plasmid. b. AAV9-SeHed virus was injected into mice by tail vein injection (5×10 11 GC per mouse). Four weeks later, mice organs (heart, liver, muscle, intestine (small intestine and colon), epididymis, Fat, brain, spleen, kidney and lung) were separated, and the EGFP signals of tissues above were detected by living imaging. c. The co-localization of mCat and COX IV in liver was performed by confocal microscopy.  Representative images of western blots for p16, p65 and p-p65 in lung. d. The senescence associated inflammatory factors mRNA levels of liver and lung were analyzed by qRT-PCR in progeria and metabolic disorder model (n=5-6/group). All data were shown as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. Figure S9. SeHed prevents HFD-induced obesity and metabolic dysfunction. a. Mice weight changes of SD, HFD and SeHed group (n=5-6/group). b. Weight of mice after feeding SD or HFD for 16 weeks (n=5-6/group). c. Fasting blood glucose levels (n=5-6/group). d. Glucose tolerance test and statistics of area under the curve (AUC) (n=5-6/group). e. Insulin tolerance test and statistics of area under the curve (AUC) (n=5-6/group). f. Mice epididymal fat index (epididymal fat/body; g/g) (n=5-6/group). g.
Representative images of HE staining and Oil Red O staining in liver (n=5-6/group).
Representative images of myotubes formation in H9C2 cells cultured in medium with 2% HS plus retinoic acid, and followed by NAC (1 mM and 3 mM) and MT (0.5 μM and 1μM) treatment for 6 days. Scale bars, 50 μm. d. Representative images of western blots for myogenin in (c). e. Representative images of neurite in PC12 cells cultured in DMEM medium with 2% HS plus Nerve Growth Factor (NGF, 50 ng/ml) for 3 days.
Scale bars, 50 μm. f. Intracellular PAO1 level was detected by Colony formation assay in Raw264.7 cells pre-treated with NAC or MT for 2 hours and then cells were infected by PAO1 for 1 hour. g. Intracellular PAO1 level was detected by colony formation assay in Raw264.7 cells pre-treated with NAC or MT for 2 hours, and then cells were infected by PAO1 for 6 hours. h. The PAO1 level in vivo was detected by living imaging in control group and NAC group (IP, 200 mg/kg; n=4-5/group) infected PAO1 for 3 hours and 8 hours. i. PAO1 level in liver and lung (n=4-5/group) in (h) were detected by colony formation assay. All data were shown as mean ± SD, * P < 0.05, ** P < 0.01; NS, no significance.               Table. S1. The primers for the experiment.